PROJECTSUMMARY/ABSTRACT?PROJECT2 Manyunknownpathogenicstepsliebetweennormaltauproteininahealthyneuronandthecellular dysfunctionthatoccursasaresultoftauuptakeandaggregation.Theaimsaddresstwoemergingfacetsof thepathobiology:themechanismoftauuptakeinneuronsandastrocytesandtheeffectsoftauinclusionsona setoffunctionalcellularparameters.Tauuptakewillbestudiedusingahighlynovelandhighlycollaborative approachcalledCRISPRitoidentifyinanonbiasedmannergenesinvolvedintauuptakefollowedby functionalstudies,whichwillvalidatetheCRISPRihits.Amorefocusedapproachtouptakewillfocuson HSPGswithaGAGmicroarraypaneltoidentifyHSPGchainfeaturestowhichtaucanbindspecifically.Once tauisinsidethecellweaskwhataretheeffectsoftauinclusionsortaumutationsinhumaniPSCderived neurons.Wehavesynthesizedmultipletypesoftauaggregatesandstudiedtheiruptakeconditions.Avariety oftechniqueswillbeappliedtopinpointcellulardefectsduetotheburdenoftauinclusions.Thesetechniques fallintotwocategories?expressionsequencingandelectrophysiologicalassessment.Withregardtothe former,thestrengthofourproposalistheuseofsinglecellRNAseqtoidentifypreciselygeneexpression changesincellswithtauinclusionscomparedtoitsneighborsthatdonothavetauinclusions.Afurther strengthisthedatathatsupportsourhypothesisconcerningtheroleoftRNAintriggeringtauconformational changesthatmayleadtoaggregationaswellasaninvestigationoftheroleofcellularstressininducingtRNA cleavageandtheformationofhalftRNAsknownastiRNAs.Wehavealsohypothesizedthattaucaninduce electrophysiologicdysfunctionandoverthepastthreeyearsincollaborationwiththephysicsdepartmenthave builttoolscapableofdetectingconductiondeficits,alterationsintheactionpotentialattheaxonalinitial segmentandhyerexcitability.Thedetectionmethodsutilizeamultielectrodearray(MEA)platform,modified MEAsthatarepatternedtoconfineneurongrowthanddirectsignalingbetweenneuronalensembles,analytical toolsforspiketrains,andanautomatedcellharvestingdevice.Thedelineationoftheeffectsontaumutations andtauinclusionsonculturedcellsisacellularphenotypethatisnotwelldescribedinthefield,butwillbe necessaryinthefutureforsmallmoleculescreens.Workinginterchangeablywithseveralcellularsystemswill allowustoselecttheidealcontextforeachexperimentalquestionposedandexploremultiplefacetsina searchfortaurelatedcellularphenotypes.Thisproposalrestsonstronginteractionsamongalltheteam members.